Na/K-ATPase ligand

ABSTRACT

This process relates to a pharmaceutical composition of an Na—K-ATPase ligand which will stimulate Na/K-ATPase signaling in a pharmaceutically acceptable vehicle. In one embodiment, the composition may be used to treat a skin disorder. In another embodiment, the composition may be used to inhibit cardiac fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication: Ser. No. 60/763,783 filed Jan. 31, 2006.

FIELD OF THE INVENTION

This invention relates to a pharmaceutical composition of an Na/K-ATPaseligand which will stimulate Na/K-ATPase signaling in a pharmaceuticallyor cosmetically acceptable vehicle. In one embodiment, the compositionmay be used to prevent the development of and treat a skin disorder. Inanother embodiment, the composition may be used to inhibit tissuefibrosis. We also have found this invention useful for the treatment ofhypertension and wound closure.

BACKGROUND OF THE INVENTION

Compounds can be used as agents through topical or systemic application.A preparation for this purpose can include a carrier, a protectant, anantioxidant (such as vitamin C or E), and other pharmaceutical andpharmacological agents. It is also expected that such compounds can beused in a delivery system (oral, local application, injection orimplantation) involving molecular recognition through which thecompounds are delivered to target site. Such a delivery system mayinvolve, among other methods, liposome techniques or immunologicaldevices. Natural or synthetic chemicals that can modulate the productionor cellular action of receptors and macromolecules are useful in thetreatment of abnormalities such as skin diseases.

Over the past decades numerous investigators have devoted significanteffort to the study of extracts of mammalian tissue and fluids in orderto identify and confirm the existence of factors that may be involved inthe regulation of Na.sup.+, K.sup.+-ATPase enzyme system. At present,considerable evidence has been produced supporting the existence of suchan endogenous factor or family of factors that is believed to inhibitthe Na.sup.+, K.sup.+-ATPase enzyme system. Moreover, these inhibitoryproperties implicate the involvement of such factors in severalphysiological roles. However, in spite of the extensive data produced bythese early investigators, considerable controversy exists with respectto their mechanisms of action, thus the physiological significance ofsuch factors.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to the utilization of certaincompounds for the control of certain disorders. We use a pharmaceuticalcomposition, comprising: a pharmaceutically effective amount of at leastone Na/K-ATPase ligand which will stimulate Na/K-ATPase signaling in apharmaceutically acceptable vehicle.

The composition induces the interaction of the signaling Na/K-ATPasewith lipids, protein kinases, phosphatases, ion channels, transporters,and other soluble and membrane proteins to form various signalingcomplexes termed Na/K-ATPase signalosomes. The process maintains normalskin structure and function. Thus, administration of an effective dosethe invented pharmaceutical composition to the subject prevents thedevelopment of and treats a skin disorder in a subject in need of suchprevention and treatment. The process includes the step of enhancingskin fibroblast collagen production by topical or injectedadministration of the pharmaceutical composition to prevent or reverseaging related loss of skin tone. The process also may include the stepsof using the pharmaceutical composition as a topical or systemicenhancement to wound closure.

We have demonstrated that the Na/K-ATPase interacts with differentlipids and proteins. These interactions result in the formation ofmultiple functional complexes that constitute the Na/K-ATPasesignalosome. The realization that the Na/K-ATPase can regulate manyimportant cellular functions and transmit the signal of CTS independentof its pumping function has promoted us to define molecular compositionsof the Na/K-ATPase signalosome and the molecular mechanisms by whichthis signalosome functions. These studies have provided us with severalnew targets for molecular interventions of the cellular function, thusnovel therapeutics and diagnostics. In one embodiment, we present one ofthose applications to prevent or reverse aging related loss of skin toneas well as to accelerate wound healing.

We discovered a relationship between high circulating levels of thecardiotonic steroid, marinobufagenin (MBG), and cardiac fibrosis inexperimental renal failure induced by partial nephrectomy (PNx). Inshort, we observed that PNx animals had substantial cardiac fibrosis.This fibrosis could also be induced by administration of MBG to achievesimilar blood levels. The fibrosis could be attenuated by immunizinganimals against MBG prior to PNx surgery. Representativeimmunohistochemistry images are shown below for illustration.

Other objects and advantages of the present invention will becomeapparent to those skilled in the art upon a review of the followingdetailed description of the preferred embodiments and the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the pathway of Na/K-ATPase signalosomes.

FIG. 2 shows the effect of marinobufagenin (MBG) on systolic BP.

FIG. 3 shows the effect of MGB on renal fibrosis.

FIG. 4 shows the effect of MGB accelerates wound healing.

FIGS. 5( a) and 5(b) show the effect of MGB on procollagen expression.

FIG. 6( a) shows dP/dt data.

FIG. 6( b) shows dP/dt data.

FIG. 6( c) shows LVEDP data.

FIG. 6( d) shows MSEC data.

FIG. 7( a) shows HW/BW data.

FIG. 7( b) shows procollagen expression demonstrated with Western blot.

FIG. 7( c) shows procollagen expression demonstrated with Western blot.

FIG. 7( d) shows procollagen expression demonstrated with Western blot.

FIG. 7( e) shows procollagen expression demonstrated with Western blot.

FIG. 7( f) shows Na/K-ATPase expression demonstrated with Western blot.

FIG. 7( g) shows SERCA expression demonstrated with Western blot.

FIG. 7( h) shows SERCA expression demonstrated with Western blot.

FIG. 8( a) shows Fibrosis-score data.

FIG. 8( b) shows Fibrosis data.

FIG. 8( c) shows Fibronectin Expression data.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the composition induces the formation of varioussignaling complexes termed Na/K-ATPase signalosomes. In anotherembodiment, the pharmaceutical composition is a pharmacologicallyeffective amount of at least one inhibitor of Na/K-ATPase signaling in apharmaceutically acceptable vehicle. The composition functions as aninhibitor of signal transduction through the Na/K-ATPase. Preferably,the treating of a skin disorder in a subject in need of such treatmentcomprises the step of administrating to the subject of an effectivetherapeutic amount of the pharmaceutical composition.

More specifically, the treatment includes the step of using thepharmaceutical composition as a topical or injected tool to reverse orprevent excessive dermal scar formation. In another embodiment, thetreating is inhibiting cardiac fibrosis in a subject in need of suchtreatment comprising the step of administering to the subject aneffective therapeutic amount of pharmaceutical composition. Thepharmaceutical composition may be in a dosage form selected from thegroup consisting of tablet, pill, suspension tablet, powder, lozenge,sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol,ointment, soft gelatin capsule, and hard gelatin capsule, suppositorycreams, lotions, solutions, gels and pastes.

The Na/K-ATPase ligands include but not limited to a group of chemicalsgenerically called cardiotonic steroids (e.g. cardenolides andbufadienolides) that are derived from either plants or animals orsemi-synthesized. The inhibitors include but not limited to those thatdisrupt the Na/K-ATPase interaction with its signaling partners such adEGF receptor. Src kinase and caveolin-1. The subjects in need oftreatment would include (but not limited to) those with systemicfibrosing conditions such as Scleroderma as well as those with localizedfibrosing conditions such as liver cirrhosis due to viral or alcoholichepatitis, progressive cardiac failure associated with renal diseaseand/or atherosclerosis as well as progressive renal disease fromglomerlonephritis, diabetes and hypertension.

The Na/K-ATPase belongs to the family of P-type ATPases that areessential for an organism to convert ATP into electric and chemicalgradients across the membranes. The Na/K-ATPase expresses in almost allmammalian cells and pumps Na+ and K+ across cell membrane using theenergy generated through hydrolysis of ATP. During the last few years,our laboratories have obtained evidence that the Na/K-ATPases alsofunctions as an important cellular signal transducer. We now suggestthat there are at least two separate pools of the Na/K-ATPase, onefunctions as an ion pump while the other engages in interaction withlipids, protein kinases, phosphatases, ion channels, transporters, andother soluble and membrane proteins to form various signaling complexestermed Na/K-ATPase signalosomes.

FIG. 1 is a schematic depicting sodium pump signaling in cardiacmyocytes. In the presence of a cardiotonic steroid, Na/K-ATPase isconverted to a signal transducer, which complexes with Src and theepidermal growth factor receptor. A signal cascade is initiated, whichdepends on Ras and results in the generation of reactive oxygen species(ROS) and activation of ERK. This, in turn, leads to altered geneexpression, including decreases in SERCA expression, as well asalterations in calcium cycling.

FIG. 2 shows MBG produces functional and anatomic changes consistentwith cardiac hypertrophy. (a) Systolic BP 4 weeks after sham operation(Sham, n=8), partial nephrectomy (PNx, n=B), MBG infusion (MBG, n=10),or immunization against MBG before partial nephrectomy (PNx-IM, n=8).(b) Representative mode echocardiograms in the 4 groups of rats. (c)Posterior wall thickness, (d) left ventricular end diastolic diameter,(e) left ventricular end systolic diameter, and (f) FS 4 weeks afterSham (n=8) PNx (n=10), MBG (n=9), or PNx-IM (n=16). *P<0.05 vs PNx;##P<0.01 vs. PNx.

FIG. 3 shows the effect of MBG on renal fibrosis.

FIG. 4 shows the effect of MBG accelerates wound healing.

FIGS. 5( a) and (b) show the effect of MBG on procollagen expression.

FIGS. 6 (a), (b), (c) and (d) MBG produces hemodynamic changesconsistent with diastolic dysfunction. (a) Maximal rate of pressurechange (+dP/dt), (b) ratio of +dP/dt tominimal rate of pressure change(i.e., most negative rate of pressure change, −dP/dt), (c) leftventricular end diastolic pressure (LVEDP), and (d) time constant ofisovolumic relaxation four weeks after sham operation (Sham, n=14),partial nephrectomy (PNx, n=15), MBG infusion (MBG, n=12), orimmunization against MBG before partial nephrectomy (PNx-IM, n=14).*P<0.05 vs. Sham; **P<0.01 vs Sham; #P<0.05 vs. PNX; ##P<0.01 vs. PNx.

FIG. 7( a) to (h) MBG produces changes in cardiac morphology and proteinexpression consistent with experimental uremia. (a) Heart weight/bodyweight (HW/BW) ratio 4 weeks after sham operation (Sham, n=18), partialnephrectomy (PNx, n=20), MBG infusion (MBG, n=20), or immunizationagainst MBG before partial nephrectomy (PNx-IM, n=18). (b) Extracellularsignal-related kinase (ERK-1, p44) activation in the left ventricularcardiac homogenate 4 weeks after Sham (n=15), PNx (n=14), MBG (n=7), orPNx-IM (n=7). Gels were loaded with 50-μg left ventricle homogenateprotein. Representative active and total ERK blots shown. (c) Src (SrcpY⁴¹⁸) activation in the left ventricular cardiac homogenate 4 weeksafter Sham (n=15), PNx (n=13), MBG (n=10), or PNx-IM (n=6). Gels wereloaded with 75-μg left ventricle homogenate protein. Representativeactive and total Src blots shown. (d) Skeletal muscle actin (skACT), (e)Na/K-ATPase a1, (f) Na/K-ATPase a2, and (g) SERCA2a expression 4 weeksafter Sham (n=15), PNx (n=13), MBG (n=10), or PNx-IM (n=B). Gels for dthrough g were loaded with 20 μg left ventricle homogenate protein. (h)SERCA2a enzymatic activity in the left ventricular cardiac homongenate 4weeks after Sham (n=8), PNx (n=6), MBG (n=8), or PNx-IM (n=8). Bargraphs for Western blot data summarize densitometry analysis of theblots. **P<0.01 vs. Sham, *P<0.05 vs Sham, #P<0.05 vs. PNx, ##P<0.01 vsPNx.

FIGS. 8( a) and (b) show MBG induces cardiac fibrosis. (a)Representative Masson's trichrome sections of left ventricular cardiactissue 4 weeks after sham operation (Sham), partial nephrectomy (PNx),MBG infusion (MBG), or immunization against MBG before partialnephrectomy (PNx-IM). (b) Semiquantitative grade and (c) quantitativemorphometric fibrosis scoring for trichrome slides of left ventricularcardiac sections 4 weeks after Sham (n=8), PNx (n=10), MBG (n=10), orPNx-IM (n=10). (d) Fibronectin expression and quantified data from Sham(n=9), PNx (n=9), MBG (n=9), and PNx-IM (n=9). Gels were loaded with 50μg of left ventricle homogenate protein. *P<0.05, **P<0.01 vs Sham,#P<0.05, ##P<0.01 vs. PNx.

Cardiotonic steroids (CTS) include plant-derived digitalis drugs such asdigoxin and ouabain, and vertebrate-derived aglycones such as bualin andmarinobufagen. Although CTS have been considered only as drugs sincetheir discovery, recent studies have identified both ouabain andmarinobufagenin as endogenous steroids whose production and secretionare regulated by multiple pathological or physiological stimuliincluding ACTH and angiotensin II. The effects of ouabain andmarinobufagenin on blood pressure have now been well-documented. Inaddition, we and others have shown recently that low doses of thesesteroids not only induced hypertension in rats, but also causedsignificant cardiovascular remodeling independent of their effect onblood pressure. In addition, these steroids regulate cell growth andcellular production of extracellular matrix proteins including collagen.

It is well established that CTS are specific ligands and inhibitor ofthe Na/K-ATPase. Early studies have demonstrated that CTS regulates geneexpression and cell growth. Recent work from our laboratories has madethe connection between the Na/K-ATPase-mediated signal transduction andCTS-evoked changes in cellular function, showing that the Na/K-ATPasecan transmit extracellular CTS signal via mechanisms independent ofchanges in intracellular Na+ and K+ concentrations. The signalingNA/K-ATPase resides in caveolae and forms a receptor complex with thetyrosine kinase Src. CTS such as ouabain act as agonists and provokethis receptor, resulting in tyrosine phosphorylation of the proteinsthat are either associated with or in close proximity to the signalingNa/K-ATPase/Src complex. Subsequently, this transactivates receptortyrosine kinases such as EGFR and initiates protein kinase cascades. Theidentified pathways include the activation of PI3K, the Ras/Raf/ERKs andPLC/PKC isozymes. It also increases mitochondrial production of reactiveoxygen species (ROS). Like other complex, activation of this complex byCTS induces the endocytosis of the activated complex, thus terminatingthe activated complex or targeting it to specific intracellularcompartment. Unlike some of the RTKs, the signaling Na/K-ATPase can alsofunction as a scaffold, capable of interacting with other membranetransporters and channels (FIG. 1). For instance, we have shown that thescaffolding and the kinase-regulatory capacities of the signalingNa/K-ATPase made it possible for the ouabain to induce Ca²⁺ transientsin cultured LLC-PK1 cells.

Immunohistochemistry studies of cardiac tissue obtained from rats withSham surgery, experimental renal failure (PNx), MBG supplementationthrough mini-pumps (MBG), and rats immunized against MBG prior to PNxsurgery. We noted the presence of protein (procollagen on left panel, asmooth muscle actin on right panel) (not shown). Similar pictures wereobtained with vimentin or fibronectin. Western blot data confirms visualtrends shown above (data not shown).

Based on these data, we next chose to examine whether MBG had directeffects on fribroblasts in a cell culture system. The following dataillustrate our findings to date.

Procollagen expression as a function of MBG concentration. Top panel iswestern blot, bottom panel mean+/−SEM of 10 experiments. ** p<0.01 vsControl Proline incorporation into collagen by cardiac fibroblasts isinduced by MBG in a dose-dependent manner. Data shown as mean+/−SEM of 6experiments ** p<0.01 vs Control (horizontal bar at 1).

We next examined whether skin fibroblasts lines had similar response.This was done with dermal fibroblasts kindly provided by Dr. BasharKahaleh. Using these human cells grown in culture, we noted that dermalfibroblasts had an enormous response to MBG as shown below. Themagnitude of this response is illustrated by comparison to maximal dosesof TGFbeta, the “classic” stimulus for fibrosis (see below). Inaddition, we observed that an immortalized fibroblast line respondedsimilarly to MBG, ouabain and digoxin.

Response of dermal fibroblasts to MBG as compared with TGF beta. Notethat increase in collagen expression in MBG is approximately 10 foldwith 5 or 10 nM MBG. Comparable effect of ouabain, MBG and digoxin onfibroblast cell line expression of procollagen demonstrated with Westernblot.

To sum these data, we observed that procollagen expression was markedlyincreased by cardiotonic steroids such as MBG. In other studies notshown here, we observed that the signaling through the Na/K-ATPaseappeared to be essential for the profibrotic effect. Moreover, weobserved that antagonism of signaling through this cascade by ROSscavenging, Src inhibition or prevention of EGFR transactivationprevented the induction of collagen synthesis.

Procollagen expression induced by MBG can be attenuated by Srcinhibition (PP2), non-specific tyrosine kinase inhibition (Herbimycin)or prevention of EGFR transactivation (AG178). Top panel isrepresentative Western blot, bottom panel is mean+/SEM of 6 experiments.** P<0.01 vs Control. Collagen synthesis assessed by pralineincorporation is increased by MBG. However both N-Acetyl Cysteine (NAC)and PP2 prevent this.

Next, we examined whether wound healing might be advantageously impactedby cardiotonic steroids. We grew a mouse fibroblast cell line, theSYF+Src line to confluence and make injuries by scraping with a pipettetip. We observed in this in vitro model of wound healing that 12 hourpretreatment with MBG markedly accelerated the closure of theexperimental lesion (see below).

Representative images from fibroblasts at 3, 7 and 12 hours followinginjury. Quantification of wound closure from fibroblast cultures. Datarepresents N=6 separate pairs of experiments, each of which involves 3determinations at each time point. Data shown as mean+/−SEM. **<0.01. Weproposed to enhance skin fibroblast collagen production by topical orinjected administration of cardiotonic steroids and prevent or reverseaging related loss of skin tone.

We also propose to develop a topical or systemic enhancement to woundclosure.

Finally, by antagonism of this process, we propose to develop a topicalor injected tool to reverse or prevent excessive dermal scar formation(e.g., keloids).

In another embodiment, we have observed recently that experimental renalfailure in the rat is accompanied by increases in circulatingconcentrations of the cardiotonic steroid, marinobufagenin (MBG), andsubstantial cardiac fibrosis. We performed the following studies toexamine whether MBG might directly stimulate cardiac fibroblast collagenproduction. In vivo studies were performed using the ⅚^(th) nephrectomymodel of experimental renal failure (PNx), MBG infusion (MBG), PNx afterimmunization against MBG, and concomitant PNx and adrenalectomy.Physiological measurements with a Millar catheter andimmunohistochemistry were performed. In vitro studies were then pursuedwith cultured isolated cardiac fibroblasts. We observed that PNx and MBGincreased MBG levels, blood pressure, heart size, impaired diastolicfunction, and caused cardiac fibrosis. PNx after immunization againstMBG and concomitant PNx and adrenalectomy had similar blood pressure asPNx but less cardiac hypertrophy, diastolic dysfunction, and cardiacfibrosis. MBG induced increases in procollagen-1 expression by culturedcardiac fibroblasts at 1 nM concentration. These increases inprocollagen expression were accompanied by increases in collagentranslation and increases in procollagen-1 mRNA without any demonstrableincrease in procollagen-1 protein stability. The stimulation offibroblasts with MBG could be prevented by administration of inhibitorsof tyrosine phosphorylation, Src activation, epidermal growth factorreceptor transactivation, and N-acetyl cysteine. Based on thesefindings, we propose that MBG directly induces increases in collagenexpression by fibroblasts, and we suggest that this may be important inthe cardiac fibrosis seen with experimental renal failure.

We have demonstrated previously that the cardiotonic steroidmarinobufagenin (MBG), signaling through the Na/K-ATPase, is directlyresponsible for many features of experimental uremic cardiomyopathyinduced by partial nephrectomy (PNx) in the rat. Specifically, we notedthat both rats subject to PNx, as well as rats given MBG supplementationby minipump, developed considerable cardiac hypertrophy and fibrosis by4 weeks, whereas rats immunized against MBG and subsequently subjectedto PNx had attenuation of these changes. From these data, we formulatedthe hypothesis that MBG might directly induce cardiac fibroblasts toproduce collagen, thus producing much of the cardiac fibrosis seen withexperimental renal failure. To test this hypothesis and to determine themolecular basis by which this occurred, the following studies wereperformed.

EXAMPLES Methods

Animals

Male, Sprague-Dawley rats were used for all of the studies. All of theanimal experimentation described in the article was conducted inaccordance with the National Institutes of Health Guide for the Care anduse of Laboratory Animals using protocols approved by the MedicalUniversity of Ohio Institutional Animal Use and Care Committee.

Experimental Groups

Briefly, Sprague-Dawley rats weighing≈250 g at the time of surgery weresubjected to either sham surgery with no MBG infusion (Sham), shamsurgery with placement of a minipump infusing MBG at 10 μg/kg per day(MBG), PNx, and PNx after immunization against MBG (PNx-IM). MBG ofextremely high purity (>99%) was isolated from the venom of Bufa marinusby Kennedy et al. In addition to these maneuvers, a group of PNx animalswas subjected to adrenalectomy as well (PNx-ADx).

The heart weight normalized to body weight, left ventricularhemodynamics (e.g. τ value, slope of regression line fit to enddiastolic pressure versus end diastolic volume generated by inferiorvena cava occlusions, all determined with a Millar catheter), plasma[MBG] (determined after extraction on a C-18 column using DELPHIA asdescribed previously), aldosterone (determined with ELISA kit 10004377,Cayman Chemical) and cardiac immunohistochemistry (vida infra) wereassessed 4 weeks after surgery.

Isolated Cardiac Fibroblasts

Preparation of adult rat cardiac fibroblasts was performed as describedpreviously by Brilla et al. with modifications.

Western Blot Analysis

Western blot analysis was performed on protein isolated from tissuehomogenates, cell culture whole cell lysates, or nuclear extracts asdescribed previously.

Collagen Synthesis

Collagen synthesis rates were determined by the method of Nishida et al.with modification.

Quantitative Measurement of Collagen-1 mRNA

Standardized RT-PCR was used to measure gene expression, with GAPDHtranscript used as the housekeeping gene, as reported previously.

Results

Effect of Experimental Renal Failure and MBG on Blood Pressure, CardiacHyemodynamics, and Fibrosis

In the current in vivo studies we observed that MBG levels wereincreased in PNx- and MBG-treated rats compared with sham-operatedcontrols. We also saw that both PNx and MBG rats had higher systolicblood pressure than controls and that PNx-IM rats had statisticallysimilar systolic blood pressure values as seen with PNx. Using theMillar pressure/volume sensor catheter rather than echocardiography inour previous report, we observed that PNx induced decreases in endsystolic volume and end diastolic volume, as well as increased ejectionfraction compared with sham-operated controls. The end systolic volumeand end diastolic volume were greater, and the ejection factor valueswere lower in PNX-IM as compared with PNx. Active relaxation assessedwas found to be impaired by both PNx and MBG compared with sham-operatedcontrols, with PNX-IM showing lower values than PNx. Using pressurevolume loops generated during vena cava occlusions, we noted that theend diastolic pressure volume relationship (an inverse measurement ofpassive compliance) was increased in PNx- and MBG-treated animalscompared with controls, whereas PNx rats ha a lower end diastolicpressure volume relationship than PNx. Both PNx and MBG treatmentincreased the heart weight/body weight ratio compared with sham-operatedcontrols, whereas PNx-IM animals had lower values than PNx. Examiningthe ventricular myocyte cross-sectional area determined on trichromeimages, we noted that PNx and MBG infusion both induced markedincreases, whereas the myocyte cross-sectional area in PNx-IM wasconsiderably smaller than that seen with PNx alone.

Effects of PNx, MBG, and PNx-IM on Hemodynamics and Plasma MBG

Effects of PNx, MBG, and PNx-IM on Hemodynamics and Plasma MBGMeasurement Sham PNx MBG PNx-IM PNx-ADx Plasma MBG, pmol/L 227 ± 27 527± 36 284 ± 47  396 ± 65  325 ± 65 Plasma aldosterone, pg/mL 184 ± 322012 ± 320 205 ± 35  2492 ± 493  228 ± 65 Tall cuff measurements Heartrate, bpm 367 ± 7  388 ± 9  367 ± 9   380 ± 6 365 ± 7 Systolic bloodpressure, mm Hg 102 ± 2  197 ± 6  136 ± 4   180 ± 9 193 ± 6 Ventricularhemodynamics End systolic volume, μL 70 ± 4 35 ± 4 60 ± 6   68 ± 9   56± 5 End diastolic volume, μL 190 ± 11 151 ± 10 162 ± 14  188 ± 14  185 ±14 Ejection fraction, % 73 ± 1 79 ± 2  68 ± 2*  71 ± 1  72 ± 2 τ, ms10.0 ± 0.3 14.5 ± 0.9  11.3 ± 0.3*  31 ± 3*  38 ± 4 EDPVRX1000, mm Hg/μL24 ± 2 52 ± 4 41 ± 6  31 ± 3*  38 ± 4 Heart weight/body weight ratio, 2.5 ± 0.1  3.6 ± 0.2  2.8 ± 0.1*  2.9 ± 0.1   3.3 ± 0.1 g/kg Analyseswere performed 4 weeks after sham operation (Sham = 20), partialnephrectomy (PNx, n = 20), IBG Infusion (MBG, n = 20), or immunizationagainst MBG prior to partial nephrectomy (PNx-IM, n = 20). Resultsreported as mean ± SEM.

Analyzing the immunohistochemistry results, heart tissues from ratssubjected to MBG and PNx showed marked increases in collagen-1 and asmooth muscle acting staining. Immunization against MBG attenuated theseincreases. Western blot analysis confirmed that PNx and MBG had 2 to 2.5times the expression of procollagin-1 and a smooth muscle actin seenwith sham-operated controls, whereas PNx-IM expression of bothprocollagen-1 and a smooth muscle actin was substantially less than thatseen with PNx.

To determine the molecular mechanism underlying this fibrosis, weexamined the expression of several proteins important in fibroplastactivation. Specifically, we examined tissue levels of transforminggrowth factor (TGF)-β, Smad 2/3, and Smad 4, as well as pSmad 2/3. Wedid not detect significant differences among the experimental groups inthe cardiac expression of these proteins.

A separate group of animals (N=11) was also subjected to PNx-ADx withphysiological replacement of glucocorticoids and aldosterone. Theseanimals developed a similar degree of hypertension compared with PNx butwere noted to have much lower plasma MBG and aldosterone levels, as wellas substantially lower heart/weight body weight ratio compared with PNxalone (Table). Moreover, these animals subjected to PNx-ADx had almostno evidence for cardiac fibrosis based on trichrome staining orimmunohistochemistry staining for collagen-1 or a smooth muscle action.

Effect of Cardiotonic Steroids on Fibroblast Collagen Expression

To further examine the molecular basis of this cardiac fibrosis,isolated cardiac fibroblasts were subjected to increase doses of MBG(10⁻¹⁰, 10⁻⁹, and 10⁻⁸ M). After 24 hours of exposure to 10⁻⁹ and 10⁻⁸ MMBG, procollagen content determined by Western blot was increased.about.2 fold (both P<0.01; FIG. 5 a). This phenomenon was not specificfor MGB; other cardiotonic steroids also induced similar increases inprocollagen content. Of interest, the threshold for effect for MBGseemed to be between 10⁻¹⁰ and 10⁻⁹ M, whereas for ouabain, whichcirculates at similar concentrations in uremic rats, the threshold wasz10 times higher (i.e., between 10⁻⁹ and 10⁻⁸ M). For both MBG andouabain, the threshold for inducing collagen expression was log unitsbelow the doses necessary for detectable effects on ⁸⁶Rb uptake in thesecells. In parallel studies examining radiolabeled praline incorporationinto collagen, we observed that 10⁻⁹ and 10⁻⁸ M MBG induced significantincreases in both proline incorporation into total protein, both matrixand supernatant. Using collagenase digestion, we observed that the vastmajority of the proline incorporation was into collagen. Usingstandardized RT-PCR, we observed a doubling of mRNA for collagen-1 at 24hours in response to 10 nM of MBG. However, we did not detect anyincreases in procollagen stability (determined by examiningprocollagen-1 expression after exposure to cycloheximide) in response tothis concentration of MBG.

Effect of Inhibition of Na/K-ATPase Signaling on MBG-Stimulated CollagenExpression

To examine whether cardiotronic steroids induced collagen synthesis bysignaling through the Na/K-ATPase, we performed the following studies.First, we used pharmacological antagonism at several steps in theNa/K-ATPase cascade. Specifically, we used pharmacological antagonism ofSrc activation with PP2, nonspecific tyrosine kinase inhibition withherbimycin, inhibition of EGFR transactivation with AG1478, andnonspecific antioxidant administration with N-acetyle cysteine. Each ofthese maneuvers prevented MBG stimulation of collagen synthesis. toconfirm these data, we also examined radiolabeled praline incorporationin response to MBG in the presence and absence of either PP2 or N-acetylcysteine. As was the case for procollagen expression, both PP2 andN-acetyl cysteine prevented increases in praline incorporation intocollagen in the primary fibroblast cultures. Next we performed studiesin the SYF and SYF+ cells (details available in the online supplement).SYF+ cells responded to MBG and ouabain in a very similar way as theprimary cardiac fibroblast cultures with respect to upregulation ofprocollagen expression, whereas the SYF cells had essentially noresponse to either MBG or ouabain.

Relationship Between TGF-β and MBG-Stimulated Collagen Production

To further examine the molecular mechanisms by which cardiotonicsteroids induce collagen production in fibroblasts, we examined theeffects of MBG on TGF-β expression, as well as the expression of Smad2/3, Smad 4, and pSmad 2/3. As was the case for the in vivo experimentsdescribed earlier, we did not observe significant changes in TGF-β, Smad2/3, Smad 4, or pSmad 2/3 expression in vitro. Next, we examined whetherTGF-β induced collagen production and whether there was synergismbetween TGF-β and MBG. In the primary cultured cells, we saw similareffects of TGF-β (5 ng/mL) on procollagen expression as observed withcardiotonic steroids; however, we did not note any synergism betweenTGF-β (5 ng/mL) and MBG (10 nM). However, it is important to point outthat we never completely serum starve the primary cultures, and becauseserum is always present, some TGF-β is always present.

To address this further, we also examined the effect of the TGF-βreceptor antagonist, SB431542, on MBG stimulated collagen production.Interestingly, SB431542 at 100-μmol/L concentration did not educeprocollagen expression below baseline on our Western blots but diddecrease radiolabeled proline incorporation below that seen with controlcells. the SB431542 completely blocked both TGF-β and MBG (10 nM)stimulation of collagen expression and radiolabeled pralineincorporation.

Cardiac fibrosis is an important component of many cardiomyopathies, andit is a very characteristic component of uremic cardiomyopathy. Ourgroup and others have observed that MBG and other cardiotonic steroidsinduce a signal transduction cascade through the plasmalemmalNa/K-ATPase residing in caveolae, which results in activation of Src,transactivation of the EGFR, generation of reactive oxygen species, and,ultimately, activation of p42/44 mitrogen-activated protein kinase.Interestingly, a number of clinical situations associated with cardiacfibrosis other than renal failure are associated with increasedcirculating concentrations of cardiotonic steroids (e.g., hypertension,primary hyperaldosteronism, and congestive heart failure). Although itis preliminary to discuss the possible relevance of our findings tocardiomyopathies other than renal failure, we should point out thatFerrandi et al. have observed that antagonism of endogenous cardiotonicsteroids with PST 2238 ameliorates hypertension, as well as cardiachypertrophy in Milan hypertensive rats.

In the current study, we confirmed that PNx and MBG treatment inducesimilar but not identical phenotypic changes in hemodynamics, andcardiac morphology. It is quite likely that some factors other than MBGcontribute to the phenotypic changes seen in PNx. That said, both PNx-IMand PNx-ADx, which reduce circulating MBG, substantially attenuate thecardiac functional and morphological changes without significantlyaffecting blood pressure. We should point out that experiments in thePNx-ADx model were performed because we reasoned that as adrenal cellsgrown in culture seem to make MBG, it was likely that this procedurewould lower the circulating levels of this hormone. However, whereas ourdata in the PNx-ADx animals support the concept that the adrenal glandis the major (but not the only) site of MBG production in vivo, it isalso possible that other hormones made in the adrenal gland modulate MBGproduction elsewhere. Further work will be necessary to clarify exactlywhere MBG is produced under normal and pathological conditions.

With these findings implicating MBG in the pathogenesis of cardiacfibrosis, we were particularly interested in the molecular mechanismsunderlying the fibrosis. Interestingly, evidence for increases in TGF-βor signaling through the Smad proteins was not evident. We stress thatthese data do not exclude a role for TGF-β in this process, becauseearlier increases in these proteins, translocation of the Smads, and/ora permissive role for signaling through this pathway (vida infra) couldcertainly be present.

Based on these in vivo data, we pursued studies in isolated cardiacfibroblasts. We observed that MBG in physiological concentrationsdirectly stimulated the fibroblasts to produce more collagen. Thisincrease in collagen production was also observed with other cardiotonicsteroids, although the threshold concentration seemed to be ≈1 log untilowe for MBG than for ouabain. We emphasize that the concentration ofboth MBG and ouabain necessary to stimulate collagen expression waslower for both substances than that needed to appreciably inhibit Rbuptake. Further evidence for this phenomenon being dependent onsignaling through the Na/K-ATPase was that this increase was preventedby reactive oxygen species scavenging, antagonism, or knockout of Src,as well as prevention of EGFR transactivation, maneuvers that we havedemonstrated previously to block signal transduction through theNa/K-ATPase signalosome. We also observed that the increases in collagenproduction were associated with increases in praline incorporation, aswell as increases in mRNA for collagen-1. No increase in procollagen-1stability could be demonstrated in response to MBG.

Although increases in TGF-β or the Smad proteins were also absent in thefibroblasts treated with MBG, it is important to note that thefibroblasts that we studied were never truly serum starved. Thefibroblasts were exposed to ≧0.12 ng/mL of TGF-β even when cultured inthe serum-depleted (1% FBS) medium. This may, in part, explain whySB431542 was to effective in preventing MBG-stimulated collagenproduction. Working with a similar preparation, Lijnen and Petrov notedthat long incubations (48 hours) and high concentrations of TGF-β (15ng/mL) were necessary to induce maximal (2 times) increases in collagenproduction. We should also note that TGF-β blockade with SB431542actually decreased proline incorporation below baseline, even in thesetting of MBG synthesis, although this same pharmacological maneuveronly reduced procollagen expression to baseline when measured withWestern blot. We suspect that other mechanisms of regulation of collagensynthesis (e.g. procollagen stability) might come into play when theTGF-β pathway is interrupted, although we did not explore this pointfurther in the current studies. On balance, our data argue, albeitpreliminarily, against a major role for TGF-β or upregulation of Smadproteins in cardiotonic steroid-induced increases in fibroblast collagenproduction.

Our data shows that, in our experimental rodent model, MBG is implicatedin the pathogenesis of the cardiac fibrosis, and the concentrations ofMBG that develop in this setting, as well as other cardiotonic steroids,have in vitro effects that are consistent with this observation. Oneissue that immediately comes to mind is whether the clinical use ofdigitalis might have similar effects. To this question, we would suggestthe following possibilities. First, it may be that the freeconcentrations of digoxin that occur in vivo are not sufficient toinduce substantial cardiac fibrosis. Total digoxin levels are typicallymaintained <2 ng/mL in patients treated with digoxin, a concentrationthat corresponds with ≈2.5-nM concentration. However, only 70% to 80% ofthe plasma digoxin is free, and the fee concentration might fall belowthe threshold level of digoxin necessary to stimulate human cardiac (orother tissue) fibroblasts. Perhaps more relevant, we observed a fairlyflat dose-response curve to MBG and ouabain with respect to stimulationof fibroblast collagen production once the threshold for an effect wasreached. We suggest that in the setting of heart failure, a conditionknown to have associated increases in MBG and other cardiotonicsteroids, the addition of digoxin at therapeutic doses might not have adetectable effect. Finally, we would point out that a systemicexamination of whether digoxin induces or influences cardiac fibrosis inhumans has not been thoroughly investigated, although the clinicalefficacy of this agent in treating congestive heart failure has beenextensively examined. It is important to note that the rate at whichhumans develop cardiac fibrosis seems to be considerably slower thanthat seen with rodents, which might further obfuscate whether digoxinhas profibrotic effects in clinical subjects.

In summary, we observed that concentrations of MBG similar to that whichdevelop in experimental renal failure produced increased synthesis ofcollagen in primary cardiac fibroblasts grown in culture in a mannerdependent on signaling through an Na/K-ATPase-Src-EGFR-reactive oxygenspecies signaling cascase. Should these data be confirmed in humans,this insight may provide useful therapeutic targets in clinical uremiccardiomyopathy.

CONCLUSION

Cardiac fibrosis is an important component of cardiac diseases seen in avariety of disease states. Our data in the experimental renal failuremodel suggest that cardiotonic steroids, such as MBG, may contribute ina very substantial role in the cardiac fibrosis seen in this setting.Because Increases in MBG are likely to accompany a variety of volumeexpansion states, the implications of our observations may extend toother situations complicated by cardiac fibrosis.

Other data includes a representative pressure-volume loops obtainedduring vena cava occlusion from rats subjected to sham surgery (Sham),PNx, MBG infusion (MBG), and PNx after immunization against anMBG-albumin conjugate (PNx-IM). Regression lines fit to the end systolicpressure volume relationship (ESPVR, dotted line) and the end diastolicpressure volume relationship (EDPVR, solid line). B, ventricularcross-sectional areas determined from trichrome stains of tissueobtained from Sham, PNx, MBG, and PNx-IM animals (each group: N=8animals, ≈100 measurements averaged to determine mean for each animal;data shown as the group mean±SEM using N-8). Representativeimmunohistochemistry images of cardiac tissues stained for (c)collagen-1 and (d) a smooth muscle actin (aSMA). Counterstain for both cand d was hematoxlyn. Western blot and corresponding densitometricanalysis for (e) procollagen-1 and (f) aSMA. Note that both collagen-1and aSMA staining are much more intense in the PNx and MBG groupscompared with Sham, whereas the PNx-IM staining is similar to Sham.Similarly, procollagen and aSMA expression are substantially higher inPNx and MBG animals compared with Sham, whereas immunization against MBG(PNx-IM) attenuated the changes seen with PNx. Data for e and f arederived from N=6 experiments in each group and shown as mean±SEM. Shamrefers to hearts isolated from control animals, PNx refers to PNx, MBGrefers to MBG supplemented, and PNx-IM refers to animals immunizedagainst MGB before PNx surgery. *P<0.05, **P<0.01 vs Sham, #P<0.01 vsPNx.

Representative Western blot for and quantitative densitometric data arefor (a) TGF-β1, (b) Smad 213, (c) Smad 4, and (d) pSmad 2/3. Dataderived from N=6 experiments in each group and shown as mean±SEM. Notesimilar expression of these proteins in all of the 4 experimentalgroups.

Representative Western blot for procollagen and quantitativedensitometric data obtained in response to different doses of (a) MBG(all N=10), (b) MBG 10 nM contrasted with different doses of ouabain(0.1 to 100 nM) and digoxin (10 nM; all N=8). c, ouabain sensitive Rbuptake as a function of MBG and ouabain concentration (N=4 at eachconcentration for both MBG and ouabain; data expressed as fraction ofcontrol). d, relative proline incorporation in the supernatant andmatrix, both will and without collagenase digestion (total). Each group(controls and different doses of MBG) contains N=7 replicants. Thedifference between the total and after collagenase digestion is reportedas collagen. The matrix is the sample that was obtained after removingsupernatant and scraping the culture dish. e, mRNA for collagen 1 inMBG-treated (10 nM; N=8) or control (N=8) fibroblasts. f, procollagenstability after cycloheximide treatment. Time 0 is 1 hour afterincubation with cycloheximide (20 μg/mL). Densitometric data displayedon log scale. Least-square regression line fit to control (CTL) and MBGdata. Bars on quantitative graphs represent the mean±SEM. *P<0.05 and**P<0.01 vs control.

The effects of PP2 (1 μmol/L), herbimycin (1 μmol/L), AG1478 (250 nM),and N-acetyl cysteine (2.5 mmol/L) on MBG (10 nM) stimulation ofprocollagen expression also were found. The PP2, herbimycin AG1478, andN-acetyl cysteine were administered from 2 hours before the addition ofMBG and continued throughout the 24 hours of MBG incubation (total of 26hours). Each bar represents the mean±SEM of n=8 experiments. b, effectsof PP2 (1 μmol/L) and N-acetyle cysteine (2.5 mmol/L) on MBG (10nM)-stimulated proline incorporation into collagen. Again, the PP2 anN-acetyl cysteine were added 2 hours before exposure to MBG. Each barrepresents the mean±SEM of N=5 experiments. c, effects of MBG 10 nM onprocollagen content in SYF and SYF+ cells. Representative Western blotsare shown above quantitative data. SYF blots loaded with 15 μg ofprotein and SYF+blots loaded with 10 μg of protein. Each bar representsthe mean±SEM of N-6 determinations in each group. **P<01.10 vs. control.

The effect of 24 hours of MBG (1 and 10 nM) on TGF-β, Smad 2/3, Smad 4,and pSmad 2/3 expression determined by Western blot. b and c, effects of24 hours of MBG (10 nM), TGF-β (5 ng/mL), and the TGF-β receptorantagonist SB431542 (100 (1 μmol/L) on procollagen-1 expression (Westernblot) and radiolabeled proline incorporation, respectively also werecarried out. SB431542 was added 2 hours before exposure to either TGF-βor MBG (total of 26-hour exposure). Each bar represents the mean±SEM of6 to 8 determinations. *P<0.05, **P<0.01 vs. control.

The above detailed description of the present invention is given forexplanatory purposes. It will be apparent to those skilled in the artthat numerous changes and modifications can be made without departingfrom the scope of the invention. Accordingly, the whole of the foregoingdescription is to be construed in an illustrative and not a limitativesense, the scope of the invention being defined solely by the appendedclaims.

We claim:
 1. A method of treating a skin disorder in a subject in needof such treatment comprising the step of administering to the subject aneffective therapeutic amount of a composition comprising at least oneNa/K-ATPase ligand selected from marinobufagenin (MBG) and digoxin in anamount effective to promote skin cell growth and to enhance skinfibroblast collagen production, wherein the effective amount is fromabout 0.1 to about 10 nM, and wherein the skin disorder is aging relatedloss of skin tone.
 2. A method according to claim 1, including the stepof enhancing skin fibroblast collagen production by topical or injectedadministration of the pharmaceutical composition to aging related lossof skin tone.
 3. The method of claim 1, wherein the composition is in adosage form selected from the group consisting of tablet, pill,suspension tablet, powder, lozenge, sachet, cachet, elixir, suspension,emulsion, solution, syrup, aerosol, ointment, soft gelatin capsule, andhard gelatin capsule, suppository, creams, lotions, solutions, gels andpastes.
 4. The method of claim 1, wherein the composition is in aninjectable form.
 5. The method of claim 1, wherein the composition isadministered in an amount not greater than 10 nM.
 6. The method of claim1, wherein the composition is administered in an amount not greater than1 nM.
 7. The method of claim 1, wherein the composition is administeredfor at least about 5 hours.
 8. The method of claim 1, comprisingadministering the composition topically.
 9. The method of claim 1,wherein the composition is administered as a daily unit dose of fromabout 1 nM to about 10 nM for at least 5 hours.
 10. A method of treatinga skin disorder in a subject in need of such treatment comprising thestep of administering to the subject an effective therapeutic amount ofa composition comprising the Na/K-ATPase ligand digoxin in an amounteffective to promote skin cell growth and to enhance skin fibroblastcollagen production, wherein the effective amount is not greater thanfrom about 1 to about 10 nM, and wherein the skin disorder is agingrelated loss of skin tone.
 11. A method according to claim 10, includingthe step of enhancing skin fibroblast collagen production by topical orinjected administration of the pharmaceutical composition to agingrelated loss of skin tone.
 12. The method of claim 10, wherein thecomposition is in a dosage form selected from the group consisting oftablet, pill, suspension tablet, powder, lozenge, sachet, cachet,elixir, suspension, emulsion, solution, syrup, aerosol, ointment, softgelatin capsule, and hard gelatin capsule, suppository, creams, lotions,solutions, gels and pastes.
 13. The method of claim 10, wherein thecomposition is in an injectable form.
 14. The method of claim 10,wherein the effective amount ranges from about 5 nM to about 10 nM. 15.The method of claim 10, wherein the composition is administered in anamount not greater than 10 nM.
 16. The method of claim 10, wherein thecomposition is administered in an amount not greater than 1 nM.
 17. Themethod of claim 10, wherein the composition is administered for at leastabout 5 hours.
 18. The method of claim 10, comprising administering thecomposition topically.
 19. The method of claim 10, wherein thecomposition is administered as a daily unit dose of from about 1 nM toabout 10 nM for at least 5 hours.